Gel electrophoresis is widely used to separate complex mixtures of molecular species, notably proteins, nucleic acids, DNA. There are principally two methods for performing an electrophoresis process: one-dimensionally and two dimensionally. In its simplest form, one-dimensional (“1D”) gel electrophoresis typically involves: (1) placing the sample(s) to be separated along or near one edge of a separating gel slab (hereinafter referred to simply as a “gel”), (2) causing an electropheretic buffer in one well or reservoir to contact the edge where the samples are located and causing an electrophoretic buffer in a second well to contact the opposite edge of the gel, and (3) applying an electrical voltage difference (hereinafter referred to simply as a “voltage”) to electrodes immersed in each well. The application of the voltage causes an electric field to be established in the gel. The electric field, in turn, causes the molecular species in each sample to migrate in the gel at different rates. The rate of migration is determined based on the molecular shape and/or charge of the molecular species, as well as the type of gel and buffer. After the migration is complete, a dying step may be performed wherein the separated molecular species are blotted onto a polyvinyiledene fluoride (“PVDF”) membrane (a nylon membrane, in the case of nucleic acids) and are then revealed by staining with dye.
In recent years, the process involved in performing gel electrophoresis has been simplified considerably through the use of commercially available pre-cast gels. Prior to this, gels were manufactured as needed in the test labs. Since gels are typically very fragile, it is necessary to protect the gels during shipment from the manufacturer to the test lab, as well as while in storage. Many of the commercially available pre-cast gels are sold sandwiched between rigid protective plastic or glass plates, while some merely have a flexible plastic backing and are stored within vacuum-sealed bags. There are also some manufacturers who supply gels in cassettes. In the field of gel electrophoresis, the term “cassette” generally refers to a rigid structure that has a gel located within it. Such cassettes not only operate to protect the gel, but also provide a convenient mechanism for transporting the gel prior to, during and after the electrophoresis process.
The steps described above for conducting the electrophoresis process can be performed in either the vertical or horizontal direction. In vertical gel electrophoresis, the gel is typically placed within a cassette that is open at both ends. Each end is in fluid communication with a different well containing a buffer. One well is typically located above the cassette and the other below. The cassette frequently serves at least as part of one wall of the upper buffer well. The cassettes that are typically used in vertical electrophoresis processes contain only the gel (i.e., no buffers or electrodes). Thus, separate wells are necessary in the vertical gel electrophoresis to provide the source for the buffer and the electrodes for providing the voltage. U.S. Pat. Nos. 5,736,022 and 6,027,628 describe conventional cassettes for use in vertical gel electrophoresis.
Heat dissipation is a major problem during gel electrophoresis. As the electric current passes through the gel, the buffer and gel begin to heat up. As the heat increases, it has a deleterious effect on the gel. If the heat is not dissipated, the gel will begin to breakdown. Accordingly, much effort has been expended in recent years to develop cooling systems that dissipate the heat generated during the process.
U.S. Pat. No. 5,888,369 describes the incorporation of an external heat exchanger in a vertical gel electrophoresis apparatus for circulation and cooling of the buffer. The apparatus accommodates cassettes that function to separate the two buffer wells. Ports are formed in one of the buffer well walls for channeling the buffer to the heat exchanger for cooling.
In horizontal gel electrophoresis, the gel is oriented predominantly in the horizontal direction. There are two general types of horizontal gel electrophoresis arrangements. In the first arrangement, the gel is placed on a slab above the two buffer wells. Each end of the gel is in contact with a porous wick that has an end located within the buffer in a buffer well. The wick conveys a sufficient amount of buffer and electrical current from the buffer well up to the gel. In the second arrangement, the gel is submerged under a thin layer of buffer, which extends from one well to the other. This is typically called “submarine” gel electrophoresis since the gel is at least partially submerged.
U.S. Patent Application Publication No. 20010037940 describes a conventional cassette for use in a horizontal gel electrophoresis apparatus. The cassette again serves to separate the two buffer wells of the apparatus. This is essentially, a horizontal adaptation of the typical vertical apparatus. The cassette includes a gel that extends into two reservoirs internal to the cassette (which are initially empty). The two reservoirs are located on opposite sides of the gel. When the cassette is inserted into the horizontal gel electrophoresis apparatus, each cassette reservoir communicates through side openings with one of the buffer wells in the apparatus. The apparatus buffer wells contain the electrodes for supplying the voltage. As such, when the apparatus buffer wells are filled with buffer, the buffer flows into the cassette reservoirs through the side openings and contacts each end of the gel. After each electrophoresis process is run, the apparatus buffer wells are emptied manually and the cassette is then removed. The cassette's reservoirs must be separately emptied.
While gel electrophoresis has become ubiquitous in the molecular biology laboratory, it has remained a laborious and time-consuming process that has largely resisted automation because of the need for human intervention at various stages. These include not only filling and emptying the buffer wells, but also removing the gel, blotting the separated molecular species onto a membrane and then staining them. Some steps have been taken to minimize or eliminate some of the labor-intensive steps. For example, U.S. Pat. Nos. 3,715,295, 3,865,712, 5,582,702 and 5,865,974 describe self-contained cassettes that include a pre-cast gel, electrodes and buffer. These cassettes only require connection to a voltage source for operation. Such self-contained cassettes are sold by Invitrogen Corporation (Carlsbad, Calif.) under the E-Gel® trademark. By their very nature, such self-contained cassettes use small amounts of buffer and carry low currents when in operation, thus eliminating the need for a dedicated cooling system.
Two-dimensional (“2D”) gel electrophoresis is an extremely powerful separation tool that is becoming an increasingly important first step in proteomic analysis. 2D gel electrophoresis typically involves: (1) a “first dimension” separation according to isoelectric point in a pH gradient gel, (2) transfer of the separated molecular species to a second gel, and (3) a “second dimension” separation according to molecular size along a direction perpendicular to that of the first separation. The need to use two different gels, and the complexity and variability of the transfer between them, make automation of 2D gel electrophoresis an even greater challenge than that of 1D gel electrophoresis.
One system for 2D separation is discussed in U.S. Pat. No. 4,443,319. The system disclosed in that patent uses a cassette that includes both the gel and the electrodes, and which is open for the admission of buffer. Provisions are made for a second gel and a second set of electrodes to be used when the cassette is used in a 2D gel electrophoresis. However, the steps involved in the disclosed system are rather cumbersome and must be performed manually.
Haber has developed a revolutionary electrophoresis technique (hereinafter referred to as the “Haber technique”) that allows separations to be performed in as little as five minutes. U.S. Pat. Nos. 3,984,298 and 4,146,454 describe the Haber technique. In addition to the short cycle time, the Haber technique utilizes low amounts of current and a small volume of buffer. Specifically, the Haber technique uses less than one milliliter of buffer in each well and is operated with currents below 0.5 mA. As such, there is no need for a cooling system. The buffers used with this technique contain conductivity suppressants. Accordingly, most of the current is carried by the molecular species being separated, rather than by ions in the buffer as in conventional gel electrophoresis. The sample is placed near the middle of the separation substrate (hereinafter referred to simply as the “substrate”). When the current is applied, some molecular species migrate toward the anode while others migrate toward the cathode. This technique is also described in N. Haber, Proc. Natl. Acad. Sci., 79, 272 (1982) and in N. Haber, Biotechnology & Histochemistry, 73, 59 (1998). An apparatus using this technique is sold by Haber Inc. (Bayonne, N.J.).
Unfortunately, the Haber technique has received little attention, perhaps because of the dearth of suitable substrates. Although Haber has used gels, cellulose and other substrate materials, most of his reported work used filter paper. The primary deficiency of filter paper is that the resulting resolution is limited by broadening from diffusion that takes place in the absence of an applied electric field.
A need therefore exists for a method, apparatus and cassette suitable for automated high-throughput electrophoretic separations on solid substrates that can take advantage of techniques that use small buffer volumes.